Plasmid Vectors and Recombinant DNA

What are the steps involved in the preparation of your recombinant plasmid with your desired DNA fragment to be cloned?

1) Cut the plasmid with restriction nuclease. 2) Cut the DNA fragment to be cloned with the same restriction enzyme (so it has complementary sticky ends). 3) Mix the two fragments together and ligate them.

Molecular biology requires the use of high quality DNA and RNA for numerous applications. What are the steps involved in extracting these materials?

1) Isolate tissue/cells. 2) Lyse the cells (to release the contents). 3) Remove proteins. 4) Remove RNA (unless you're going to use it). 5) Concentrate DNA sample.

What are the steps involved with Alkaline lysis miniprep?

1) Lyse the bacteria in a solution to release the contents. 2) All of the DNA is released and placed into a tube containing alkaline solution (denatures the DNA). 3) With the denatured DNA and proteins aggregated at the bottom of the tube, adjust the pH to neutral. 4) The plasmids will be released and float toward the top of the solution, use column purification (spin and filter) to isolate the pure plasmid DNA.

What are the 3 basic plasmid components?

1) Origin of Replication. 2) Antibiotic Resistance marker. 3) Multi-cloning site containing unique restriction enzyme sites.

How would you probe for a particular gene using an agarose plate?

1) Plate your human genomic library on an agarose plate. 2) Lay a charged membrane over the colonies (to which they will attach). 3) Lyse the cells on the membrane and add a DNA probe. 4) Further amplification using film.

What is meant by a genomic library?

A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA.

What is a cloning vector? Which problem with DNA digestion does its utility solve?

A plasmid that has been genetically engineered to perform a specific function. This is a way of sorting through all of the various fragments of DNA after cleavage with restriction enzymes.

How does Northern/Southern Blotting work?

A sponge is placed in a dish filled with alkali (salt) solution. On top of the sponge you place the agarose gel. Over the gel goes a charged membrane and on top of that place stacks of paper towels. Through capillary (wicking) action, the salt solution is drawn up through the sponge and then the gel, lifting all of the DNA from the gel and attaching to the charged membrane above it.

What is a technique used to retrieve the plasmid from the bacteria?

Alkaline Lysis Miniprep.

Once the recombinant plasmid is inserted into the DNA, what comes next?

Allow it to grow on a plate laced with a particular antibiotic so that only the bacteria cells of interest (those containing the recombinant plasmid) will be able to proliferate.

Once you have your recombinant plasmid, how does it become transformed (inserted) into bacteria?

Chemically: Making the bacteria "leaky" using chemicals and they will suck up the plasmid. Electrically: Use an electric field to push it into the bacteria.

What is EtBr?

Ethidium bromide - an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis.

What is meant by high copy number vs. low copy number in recombinant plasmids?

High copy number - high level of replication. Low copy number - low level of replication.

How does agarose gel function?

It acts like a sieve - if you push DNA through the gel in the presence of an electric field it will separate out DNA based on molecular size.

What property of DNA ensures that it will migrate during gel electrophoresis?

It is highly negatively charged - it will be attracted to the positive pole on the opposite side.

What is the medium called used to grow human fibroblasts? Why is this medium used? How do they grow?

Minimal Essential Medium (MEM). It contains all of the necessary nutrients to keep a human cell alive. The cells grow in the bottom layer - usually one layer deep.

What are plasmids?

Naturally occurring extrachromosomal pieces of (circular) DNA that are capable of self replicating.

If you have DNA or RNA run out onto a gel, how would you transfer it to a solid support if you wanted to continue to manipulate it?

Northern/Southern Blotting.

Once your bacteria cells containing your recombinant plasmid are allowed to grow on the plate, what comes next?

Place your cultures into a broth medium in the presence of antibiotics and heat it up to 37◦ in order to then reproduce it in large quantities.

What is a "sticky end"? What are they useful for and why?

Refers to the properties of the end of a molecule of DNA or a recombinant DNA molecule. The concept is important in molecular biology, especially in cloning or when subcloning inserts DNA into vector DNA.

What is meant by the term "molecular scissors"? What are they used for?

Restriction enzymes - needed for molecular biology techniques - allow for the manipulation of DNA fragments.

What is the difference between Southern and Northern blotting?

Southern Blotting - used for DNA. Northern Blotting - used for RNA.

Why might you get a smear when running a gel electrophoresis on human DNA?

The human genome contains countless restriction enzyme sites. This means that when the genome is all cut up into fragments, there will be a large mix of fragment variants instead of just a few different sizes which would create bands.

How does blue/white screening work?

The multi-cloning site of the plasmid is within the LacZ gene. LacZ, in the presence of a certain substrate, secretes an enzyme that makes blue colonies. If you disrupt this gene by adding a different DNA sequence, this throws the LacZ gene out of frame and white colonies will be produced. The white colonies are the ones with the recombinant DNA and the blue ones are the normal, unaffected plasmid DNA.

Which pieces of DNA will travel the furthest when running a gel with electrophoresis?

The smaller pieces of DNA will migrate the further in the gel than the larger pieces which get held up more in the gel.

How do restriction enzymes function?

They recognize certain DNA base pair patterns arranged in palindromic sequences and they cut at these sites.

What is blue/white screening?

This is a way of determining which colony contains your DNA fragment that you're trying to clone into the plasmid - cells transformed with vectors containing recombinant DNA will produce white colonies.

Why would you use a blotting technique? What do you do after you have the DNA on the charged membrane?

To determine if your cloning experiment was successful. Place the membrane into a solution containing a probe DNA specified to identify a particular gene of interest.

Why are antibiotics used again in the culture broth?

To ensure that the only bacteria allowed to grow in your broth are the ones containing the recombinant plasmid.

Once you have DNA fragments from processing with restriction enzymes, how can the fragments be separated?

Using agarose gel that size fractionates different sized pieces of DNA molecules.

How can cloning vectors be used to manipulate fragments of DNA?

We can place DNA pieces into the programmed plasmid in order to amplify desired regions of the genome.